**2. Current manufacturing strategies for stem cell therapy**

Current manufacturing strategies for cell therapy products are replicated from biologics manufacturing in the pharmaceutical industry. However, the processing of cells is far different from pharmaceutical proteins or vaccines. For pharmaceutical peptide production from microorganisms, the raw materials are extracted from bacteria or fungus [11, 12]. They are then separated, purified, and examined for quality assurance to meet the requirements of regulatory agencies, e.g., Food and Drug Administration (FDA), British Pharmacopeia, etc. The final products are stored or marketed in a dose-dependent manner.

Cell processing is more intensified when the pharmaceutical proteins are produced by using human, animal, or plant cells as a by-product. In this case, high quality products depend on the maintenance of high quality cells, and maintaining a sterile condition is very important. Therefore, good bioprocessing is required to optimize the production of desired proteins. After inoculating from a master cell bank, the cells are cultured for a specific period of time [13, 14]. The supernatant is then collected and the desired proteins are separated, purified, and concentrated. The isolated products then go through quality assurance to meet the criteria of the regulatory agency. Finally, the products are stored and marketed in a dose-dependent manner.

The manufacturing of stem-cell based products is not as straight forward as the production of pharmaceutical proteins or vaccines. This is because cells are the final product in stem cell therapy and are vulnerable to physical or chemical operations from isolation to delivery to patients. Cell manufacturing strategies also vary from source to source and depend on autologous or allogeneic transplantation (**Figure 1**). The major general steps are the acquisition of tissue samples and isolation of cells, initial cell purification, selection, activation and transduction, cell expansion, differentiation, washing, harvesting and formulation, filling and cryopreservation, and finally, storage and delivery to the clinics [10].

For stem-cell based products, cells are isolated from specific tissues of patients, e.g., blood, skin, etc. for autologous transplantation or can be used from cell banks for allogeneic transplantation. Heterogeneity of final products may arise from the cell isolation step because patients' tissues contain various undesired

*Current Topics in Biochemical Engineering*

worldwide day by day [6].

storage of final products.

high quality cells for treating incurable diseases [8, 9].

for autologous cell therapy applications, they are less preferable for clinical trials because of less genetic stability compared to the hESCs. However, a few clinical trials have already been started using the patient-derived hiPSCs. The Takahashi group from the Riken Center for Developmental Biology has recently conducted a clinical trial for treating wet AMD [4]. Similarly, a Takahashi from Kyoto University is conducting a clinical trial for treating Parkinson's disease by using hiPSCs [5]. A few clinical trials are also ongoing in the USA for treating different diseases like β-thalassemia, liver diseases, diabetes, etc. using hiPSCs and their use is expanding

As stem cell therapy is garnering increasing attention, a lot of clinical trials are ongoing using both hESCs and hiPSCs cells. About 6849 clinical trials and 1415 stem cell-based therapies were found based upon searches we recently performed on clinicaltrials.gov (October, 2018) [7]. However, the percentage of success is not high enough as speculated from the previous clinical trials. Among the 315 clinical trials conducted (26.0% Phase 1, 40.6% Phase 1/2, 22.5% Phase 2, 3.8% Phase 2/3, and 6.7% Phase 3), only 0.3% went to Phase 4 [3]. The low percentage of completion of clinical trials depends on various factors. One of the major factors is manufacturing practices that can provide high safety and efficacy of cell therapy products. Moreover, production cost of multiple doses also hinders the success rate of clinical trials. As cell therapy revenue exceeded multi-million dollars and has been a profitable business in recent years, but much attention is needed to produce

The production of stem cell biologics is adapted from the conventional pharmaceutical protein and vaccine production. Conventional biologics production involves the following basic steps: isolation and identification of raw materials, formulation, filling, packaging, and storage, where the total processing stops at the

*Schematic illustration of current multi-step cell manufacturing strategies in planar culture for stem cell therapy applications. Skin cells are isolated from the patient and reprogrammed to hiPSCs using viral vectors. After reprogramming, hiPSCs are stored in a master cell bank or differentiated directly in autologous cell therapy applications. In some cases like allogeneic cell therapy applications, cells are expanded in a large amount and then differentiated. After performing characterization, quality assurance, and screening for safety and efficacy,* 

*cells are delivered to hospital or stored in a cell bank for future use.*

**70**

**Figure 1.**

subpopulations. For example, in chimeric antigen receptor T-cell (CAR-T) therapy, cells are isolated from patients' blood tissue, which contains abnormal levels of inhibitory factors and regulatory cells [15, 16] because patients are treated with chemo- and radiotherapies. As a result, heterogeneity occurs in the final products, which need much attention during the cell isolation step. Cells isolated from patients need to be purified by centrifugation, magnetic-activated cell sorting (MACS), or fluorescent-activated cell sorting (FACS). Then, initial cell culture is done for selection, activation, or transduction of specific interest.

After purification, cells are expanded in plate culture or bioreactor. Based on demand, large-scale expansion is required in a sterile condition, which also requires intensive consideration because it is the rate-limiting step for commercialization of cell therapy products. The most important considerations for large-scale expansions are: operational, economic, quality and safety.

Operational design for culture systems (2D or 3D) with manual or automatic (desirable) operation is important before large-scale expansion [17]. Bioreactors are superior to plate culture for obtaining a large number of cells. Online monitoring and control of process parameters (pH, DO, pCO2, etc.) and considering the shortest possible culture time are also important parameters for operational consideration. A prediction model for medium consumption (glucose and glutamine) and toxic material production (lactic acid and ammonium) is very useful for determining medium feeding regimen. A dedicated single-use vessel is also a big operational consideration before large-scale expansion of cell-based products.

As cell-based products are costlier, economic considerations for medium, efficient cell lines and other indirect utilities are important. However, the most important consideration in large-scale expansion is product quality and safety. For this purpose, dedicated cell manufacturing facilities are required to maintain current manufacturing practices (cGMP) for high product purity and safety.

After large-scale expansion, cells are harvested by detaching them from the culture substrate using enzymatic treatment. Non-enzymatic detachment is also available by changing temperature or pH [18–20]. Aggregate culture in bioreactors may not necessarily need a detachment step for harvesting [21–25]. Next steps are washing and volume reduction, which can be done by centrifugation or tangential flow filtration on a large scale by using automated commercial devices (kSep systems and Terumo BCT).

Purified cells are formulated in a dose-dependent manner and checked for quality assurance. Quality assurance is done in three different stages: microbial contamination, chemical contamination, and quality or potency assurance. Microbial contamination is checked for bacterial, fungal, or viral contamination by sterility tests with various methods [26, 27]. The most commonly used sterility test is a 14-day incubation of cell products for bacterial and fungal contamination [28, 29]. Chemical testing includes checking for molecules accompanying the culture medium or other factors used during isolation, expansion, and storage. One commonly used chemical test is the LAL test for bacterial endotoxin. There is now an automated 15 min test for determining endotoxin in cell therapy products, which was developed following FDA regulations [30]. Other chemical testing concerns are checking for residual proteins of different origins, serum, and other harmful particles originated from cell processing.

In cell therapy products, quality is the major concern, especially because cell growth is a requirement. For that reason, a cell viability assay is done to determine live or dead cells in the product using a variety of staining methods. Colony forming unit (CFU) is also useful for determining biological activity of cell therapy products [31, 32]. Product potency is an important criterion to meet before releasing the product. For example, if a cell therapy product is applied for the chimeric antigen

**73**

*Integrated Biologics Manufacturing in Stirred-Suspension Bioreactor: A Stem Cell Perspective*

For hPSC-derived products, strict quality control is imperative before transplantation to the patients because there is high risk of oncogene transfer to patients. A clinical trial was halted in 2015 in Japan while treating AMD by autologous hiPSC-derived retinal pigmented epithelial cells because of genetic abnormality [34]. Since genetic abnormalities occur in hiPSC-derived products from reprogramming to finally differentiated cells [35], cells should be strictly screened for epigenetic signatures, karyotyping, telomerase activity, mitochondrial remodeling, etc. [36–38]. Rohani et al. summarized possible molecular cytogenetics for quality control that should be checked before releasing the final products [39]. Some of the proposed quality testings are whole-genome sequencing, single-cell genome sequencing, epigenomic analysis, and mitochondrial DNA integrity test-

receptor T (CART)-related cancer therapy, it needs to be examined for the secretion of cytotoxic cytokines (IFN-γ) and killing of target cells [33]. However, for hPSCs, the final products are differentiated cells, wherein potency should be checked via

After passing the product quality assurance, cells need to be delivered to clinics immediately or stored for future use. Cells are shipped generally to the clinics on dry ice (−78°C) or in liquid nitrogen dry shippers (−160°C) if the cells are vitrified. The mostly used technique for cell storage is cryopreservation in liquid nitrogen at −196°C which is adapted from the conventional stem cell banking [40, 41]. For cryopreservation, dimethyl sulfoxide (DMSO), glycerol, sugars, or other polymers are used. Among them, clinical grade DMSO is widely used although it is detrimental and can cause harmful effects to cells [42, 43]. Therefore, removing it from cryopreservation protocols or lowering the concentration is important. However, developing appropriate protocols for freezing and thawing is also important for high recovery of cells. Generally, slow-freezing and quick thawing is highly applicable for better recovery of cryopreserved cells [44, 45]. Since intracellular ice crystal formation is a big obstacle in cryopreservation, using ice recrystallization inhibitors is also an effective process for cryopreservation of clinical cell therapy products [46, 47]. Product delivery is also an important step to consider before administration to the patients. Since the products are carried in an environment where temperature is extreme, the container should be made with such materials that can withstand extreme low temperature and do not cause any leakage compromising the product quality [48]. For autologous cell therapy applications especially for CAR-T cell therapy, a dedicated

vessel, which can withstand extreme low temperature, is needed [49].

The conventional production of pharmaceutical proteins or other biologics consists of multiple steps from raw materials to finished products. As biologics need to maintain stringent quality control, multiple steps in production facilities compromise the product quality significantly. They also reduce productivity and become prone to human errors, which decrease product efficacy and safety. Moreover, multiple steps in cell processing consume a lot of time, which indirectly increases production cost. To overcome these drawbacks, integrated pharmaceutical production has been attempted by various pharmaceutical companies. One of the significant attempts was made by the Novartis-MIT Center for Continuous Manufacturing of pharmaceutical products to fully integrate the cell processing system [50, 51]. Another attempt was taken by Genzyme™ for continuous production of pharmaceutical recombinant protein in bioreactors, where cell culture to product isolation and purification was integrated in a single flow [52]. By using this system,

**3. Integrated biologics manufacturing in bioreactors**

*DOI: http://dx.doi.org/10.5772/intechopen.83813*

transplantation into disease models.

ing for maximizing the patient safety.

*Integrated Biologics Manufacturing in Stirred-Suspension Bioreactor: A Stem Cell Perspective DOI: http://dx.doi.org/10.5772/intechopen.83813*

receptor T (CART)-related cancer therapy, it needs to be examined for the secretion of cytotoxic cytokines (IFN-γ) and killing of target cells [33]. However, for hPSCs, the final products are differentiated cells, wherein potency should be checked via transplantation into disease models.

For hPSC-derived products, strict quality control is imperative before transplantation to the patients because there is high risk of oncogene transfer to patients. A clinical trial was halted in 2015 in Japan while treating AMD by autologous hiPSC-derived retinal pigmented epithelial cells because of genetic abnormality [34]. Since genetic abnormalities occur in hiPSC-derived products from reprogramming to finally differentiated cells [35], cells should be strictly screened for epigenetic signatures, karyotyping, telomerase activity, mitochondrial remodeling, etc. [36–38]. Rohani et al. summarized possible molecular cytogenetics for quality control that should be checked before releasing the final products [39]. Some of the proposed quality testings are whole-genome sequencing, single-cell genome sequencing, epigenomic analysis, and mitochondrial DNA integrity testing for maximizing the patient safety.

After passing the product quality assurance, cells need to be delivered to clinics immediately or stored for future use. Cells are shipped generally to the clinics on dry ice (−78°C) or in liquid nitrogen dry shippers (−160°C) if the cells are vitrified. The mostly used technique for cell storage is cryopreservation in liquid nitrogen at −196°C which is adapted from the conventional stem cell banking [40, 41]. For cryopreservation, dimethyl sulfoxide (DMSO), glycerol, sugars, or other polymers are used. Among them, clinical grade DMSO is widely used although it is detrimental and can cause harmful effects to cells [42, 43]. Therefore, removing it from cryopreservation protocols or lowering the concentration is important. However, developing appropriate protocols for freezing and thawing is also important for high recovery of cells. Generally, slow-freezing and quick thawing is highly applicable for better recovery of cryopreserved cells [44, 45]. Since intracellular ice crystal formation is a big obstacle in cryopreservation, using ice recrystallization inhibitors is also an effective process for cryopreservation of clinical cell therapy products [46, 47].

Product delivery is also an important step to consider before administration to the patients. Since the products are carried in an environment where temperature is extreme, the container should be made with such materials that can withstand extreme low temperature and do not cause any leakage compromising the product quality [48]. For autologous cell therapy applications especially for CAR-T cell therapy, a dedicated vessel, which can withstand extreme low temperature, is needed [49].

## **3. Integrated biologics manufacturing in bioreactors**

The conventional production of pharmaceutical proteins or other biologics consists of multiple steps from raw materials to finished products. As biologics need to maintain stringent quality control, multiple steps in production facilities compromise the product quality significantly. They also reduce productivity and become prone to human errors, which decrease product efficacy and safety. Moreover, multiple steps in cell processing consume a lot of time, which indirectly increases production cost. To overcome these drawbacks, integrated pharmaceutical production has been attempted by various pharmaceutical companies. One of the significant attempts was made by the Novartis-MIT Center for Continuous Manufacturing of pharmaceutical products to fully integrate the cell processing system [50, 51]. Another attempt was taken by Genzyme™ for continuous production of pharmaceutical recombinant protein in bioreactors, where cell culture to product isolation and purification was integrated in a single flow [52]. By using this system,

*Current Topics in Biochemical Engineering*

subpopulations. For example, in chimeric antigen receptor T-cell (CAR-T) therapy, cells are isolated from patients' blood tissue, which contains abnormal levels of inhibitory factors and regulatory cells [15, 16] because patients are treated with chemo- and radiotherapies. As a result, heterogeneity occurs in the final products, which need much attention during the cell isolation step. Cells isolated from patients need to be purified by centrifugation, magnetic-activated cell sorting (MACS), or fluorescent-activated cell sorting (FACS). Then, initial cell culture is

After purification, cells are expanded in plate culture or bioreactor. Based on demand, large-scale expansion is required in a sterile condition, which also requires intensive consideration because it is the rate-limiting step for commercialization of cell therapy products. The most important considerations for large-scale expansions

Operational design for culture systems (2D or 3D) with manual or automatic (desirable) operation is important before large-scale expansion [17]. Bioreactors are superior to plate culture for obtaining a large number of cells. Online monitoring and control of process parameters (pH, DO, pCO2, etc.) and considering the shortest possible culture time are also important parameters for operational consideration. A prediction model for medium consumption (glucose and glutamine) and toxic material production (lactic acid and ammonium) is very useful for determining medium feeding regimen. A dedicated single-use vessel is also a big operational

As cell-based products are costlier, economic considerations for medium, efficient cell lines and other indirect utilities are important. However, the most important consideration in large-scale expansion is product quality and safety. For this purpose, dedicated cell manufacturing facilities are required to maintain cur-

After large-scale expansion, cells are harvested by detaching them from the culture substrate using enzymatic treatment. Non-enzymatic detachment is also available by changing temperature or pH [18–20]. Aggregate culture in bioreactors may not necessarily need a detachment step for harvesting [21–25]. Next steps are washing and volume reduction, which can be done by centrifugation or tangential flow filtration on a large scale by using automated commercial devices (kSep

Purified cells are formulated in a dose-dependent manner and checked for quality assurance. Quality assurance is done in three different stages: microbial contamination, chemical contamination, and quality or potency assurance. Microbial contamination is checked for bacterial, fungal, or viral contamination by sterility tests with various methods [26, 27]. The most commonly used sterility test is a 14-day incubation of cell products for bacterial and fungal contamination [28, 29]. Chemical testing includes checking for molecules accompanying the culture medium or other factors used during isolation, expansion, and storage. One commonly used chemical test is the LAL test for bacterial endotoxin. There is now an automated 15 min test for determining endotoxin in cell therapy products, which was developed following FDA regulations [30]. Other chemical testing concerns are checking for residual proteins of different origins, serum, and other harmful

In cell therapy products, quality is the major concern, especially because cell growth is a requirement. For that reason, a cell viability assay is done to determine live or dead cells in the product using a variety of staining methods. Colony forming unit (CFU) is also useful for determining biological activity of cell therapy products [31, 32]. Product potency is an important criterion to meet before releasing the product. For example, if a cell therapy product is applied for the chimeric antigen

rent manufacturing practices (cGMP) for high product purity and safety.

done for selection, activation, or transduction of specific interest.

consideration before large-scale expansion of cell-based products.

are: operational, economic, quality and safety.

systems and Terumo BCT).

particles originated from cell processing.

**72**

they respectively reported successful production of monoclonal antibody as well as highly complex, less stable pharmaceutical protein with consistent product quality, high product output, and low cost. Process integrity is necessary for reducing cumbersome production steps and cutting cost significantly. One such integrated system developed by Johnson & Johnson has recently got FDA approval for largescale HIV drug production [53] that reduces time and cost by one third compared to the conventional batch processing.

Since biologics production for cell therapies require multiple steps, integration of all of the steps will give high product quality and safety, as well as help overcome stringent regulatory requirements. In this context, we will discuss how to integrate some important basic steps of cell manufacturing especially genetic modification, cellular reprogramming, expansion, and differentiation in bioreactors to promote a single-step approach for cell-based therapies (**Figure 2**).
